The ability to measure directly forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, the last 1-1.5 nanometers separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, and for the exclusion of small solutes and salts from polar and nonpolar macromolecular surfaces has led us to conclude that the energy associated with changes in structuring water between surfaces dominates intermolecular forces. Dense packaging DNA in eukaryotic nuclei, sperm nuclei, viruses, and bacteria is necessary for proper cellular functioning. DNA assembly by multivalent ions is a critical testing ground for understanding not only in vivo compaction of DNA but also the physics of interactions between charged molecules. If a sufficient concentration of multivalent ions is present, DNA will spontaneously assemble into an ordered array. The helices do not collapse to touching but are rather separated by 0.5-1.5 nm of solvent depending on the nature of the condensing ion. Attractive and repulsive forces balance at the equilibrium spacing. We have been able to separate the attractive and repulsive free energies at the equilibrium spacing for the several commonly used condensing agents and for a series of homologous oligo-arginine peptides. The results confirmed our previous hypotheses for hydration forces. There is a 0.25 nm decay length exponential repulsive force that is the hydration equivalent of the image charge repulsive force in electrostatics. The hydration atmosphere extending from a solvated surface stabilizes water structuring at the surface. Disruption of the atmosphere by replacing water with another surface will lower hydration energies regardless of the water structuring on the other surface. The attraction is also characterized by an exponential force but with twice the decay length (0.5 nm) of the repulsive force. Attraction results from the direct interaction of surface hydration structures. Perturbations in water structure around one surface due to the close presence of another surface can either weaken or strengthen hydration energies depending on the mutual structuring water. We postulated that the attractive force had the same exponential 0.5 nm decay length as observed previously for repulsive hydration forces, but that the force was now attractive because of correlations in complementary water structuring on apposing helices. By further investigating a set of oligo-arginine peptides of varying lengths, we determined that the magnitude of the attractive force increases with the number of charges consistent with a constant loss in entropy from correlating a single molecule regardless of charge, but a gain in interaction energy that increases with the number of charges. The 0.25 nm decay length repulsive force is only slightly dependent on the number of charges, but does depend on the chemical nature of the bound counterion. We have extended this work and are now investigating condensed arrays of protamine assembled DNA. Protamines are small (30-50 aa), arginine-rich (60-65%) peptides used to package DNA in sperm heads. Significantly, protamine-DNA forces resemble the forces seen for all other condensing ions we have measured, from simple metal ions as Mn2+ and Co3+ to the biogenic amines spermidine and spermine to the oligo-arginine peptide series. There seems to be no additional contribution from the protein character of protamines. The repulsive force, however, is significantly larger than expected from the oligo-arginine peptide measurements, suggesting that the neutral amino acids present in protamine result in an increased residual repulsion. We have confirmed this hypothesis by measuring DNA force curves with synthetic hexa-arginines that incorporate increasing numbers of neutral amino acids. Surprisingly the character of the neutral amino acid from hydrophilic serines to hydrophobic isoleucines makes little difference to the overall force magnitude. We have also measured DNA assembly forces in salmon sperm nuclei. The in vivo sperm and in vitro, reconstituted salmon protamine - DNA forces are virtually identical. These force measurements thus have direct biological application. Mammalian protamines are more complicated than piscine;two protamine species are typically present and disulphide bridges form between protamines presumably to further condense DNA. This can be easily verified by measuring DNA-DNA spacing with and without added disulphide reducing agents. We will optimize our experimental protocols with bull sperm before investigating human sperm. We can use our knowledge of DNA assembly forces to investigate the connection between DNA packaging in sperm heads, DNA damage, and male infertility. There are several reports in the literature correlating human male infertility with protamine abnormalities. Cumulative DNA damage due to reactive oxygen species is a major contributor to male infertility since DNA repair systems are absent in sperm. We hypothesize that incorrect assembly of DNA by insufficient or modified protamines will result in less tightly packaged DNA in sperm (a larger than normal distance between helices) and, consequently, a greater accessibility of oxidizing free radicals to DNA. We can test this hypothesis by direct measurements of the distance between helices in sperm of normal and infertile males and of the accumulated DNA damage. The replacement of histones by protamines in spermatogenesis occurs in several steps. Before binding to DNA, the several of the protamine serines are first phosphorylated. It is only afterwards that phosphorylated protamine- DNA complexes are dephosphorylated and the DNA fully packaged. We have examined the effect of phosphorylation of synthetic protamine-like peptides on DNA-DNA forces. As expected from previous measurements, R3SR3 is only slightly different from R6. The phosphorylated peptide, R3pSR3, however, profoundly weakens attraction, increasing the distance between DNA surfaces from 0.85 nm to 1.35 nm even in the absence of any competing salt as would be found in the cell. This level of phosphorylation mimics that found in naturally occurring protamines. This greatly weakened attraction suggests that the function of protamine phosphorylation is to prevent tight packaging of DNA and to allow phosphatase access. An orderly dephosphorylation of these modified protamines would consequently allow an orderly condensation of DNA. Incomplete dephosphorylation and, therefore, less tightly assembled DNA has been linked to infertility. We are currently phosphorylating Clupine protamine using protein kinase A. This particular protamine has three consensus PKA sequences for serine phosphorylation. We have also investigated the additivity of DNA-DNA forces with mixed ions. The force magnitudes of both the 2.5 and 5 Angstrom decay length forces vary with the kind of ion bound to DNA. This ion specificity depends on charge or water organizing ability and binding location on DNA. Bound ions may interact synergistically to maximize attraction. This knowledge is important for predicting interaction energies in real biological systems. To this end, the forces in cobalt hexamine+3, Mg2+, and putrescine2+ were separately and carefully measured and then parameterized. Forces were then measured in 1 mM Co3+ with concentrations of Mg2+ or putrescine2+ varied from 4 to 80 mM. The fraction of DNA charge neutralized with Co3+ was determined spectrophotometrically. To a good first order approximation, the observed forces were simply a linear combination of the contributions from each of the bound ions.